Nucleic acid aptamers are single-stranded DNA or RNA molecules capable of binding to small molecules, proteins, and whole cells with high affinity and selectivity. Aptamers have been widely applied both as therapeutic reagents and as promising tools for molecular diagnostics and basic research, and are commonly selected in vitro from large libraries of DNA or RNA sequences using Systematic Evolution of Ligands by Exponential Enrichment (SELEX).
It is known that the selection of aptamers having high affinities and specificities for specific proteins can be done from a large library by incorporating modified dUTP in the SELEX process. The process, however, typically requires multiple cycles (8-20 rounds) of partitioning, separation, and PCR amplification steps. As such, several attempts have been made to improve the time and resources required for the SELEX process, including attempts to achieve the selection of aptamers using a single cycle.
One such attempt, known as the capillary electrophoresis SELEX (CE-SELEX), may be used to select an aptamer for h-Ras protein in a single cycle by repeated, highly efficient separation of the protein-bound sequences from the unbound sequences. However, due to the limited injection volume of the CE-SELEX system, over 97% of the DNA sequence diversity was lost, although initial DNA library contained 2×1012 ssDNA sequences.
Another such attempt, which involves a magnetic microfluidic chip (M-SELEX) can, in principle, be applied to any protein target. The M-SELEX strategy may achieve extremely high selection stringency by exposing a very small quantity of target proteins to a DNA library during the partitioning step. This method is limited, however, to microscale sample volumes and may be subject to the risk of compromising sequence diversity. This method also requires immobilization of the target molecules onto micromagnetic beads prior to aptamer selection.
There is a need to develop a consistent and reliable single-cycle aptamer selection strategy that takes full advantage of the large DNA library diversity and this is amenable to common laboratory practice. Such a system may be capable of directly selecting aptamers for proteins on a membrane, and may not be limited by sample volumes. Such a system may further be capable of being integrated into common protein separation techniques used in biochemistry laboratories such as, gel electrophoresis and membrane blotting.
Both of Musheev, M.: Krylov, S. N. Analytica Chim. Acta 2005, 564, 91-96 and Oh, S. S.; Plakos, K.: Lou, X.; Xiao, Y.; Soh, H. T. 2010, Proc. Natl. Acad. Sci. U.S.A. 107 (32) 14053-14058 are incorporated by reference herein in their entirety.